In agriculturally important seed crops, the expression of storage protein genes directly affects the nutritional quality of the seed protein. In maize, the prolamine (zein) fraction of storage proteins comprises over 50% of the total protein in the mature seed. The zeins designated α-zein are especially abundant. The α-zein polypeptides contain extremely low levels of the essential amino acids lysine and tryptophan. Thus, maize seed protein is deficient in these amino acids because such a large proportion of the total seed storage protein is contributed by the α-zeins (Mertz et al., 1964).
The development of breeding steps to improve maize based on the manipulation of zein profile is hampered by the complexity of the zein proteins. The term “zein” encompasses a family of some 100 related proteins. Zeins can be divided into four structurally distinct types: α-zeins include proteins with molecular weights of 19,000 and 22,000 daltons; β-zeins include proteins with a molecular weight of 14,000 daltons; γ-zeins include proteins with molecular weights of 27,000 and 26,000 daltons; and δ-zeins include proteins having a molecular weight of 10,000 daltons. The α-zeins are the major zein proteins found in the endosperm of maize kernels. However, the complexity of zein proteins goes beyond these size classes. Protein sequence analyses indicates that there is microheterogenicity in zein amino acid sequences. This is in accord with isoelectric focusing analyses which show charge differences in zein proteins. Over 70 genes encoding the zein proteins have been identified (Rubenstein, 1982), and the zein genes appear to be located on at least three chromosomes. Thus, the zein proteins are encoded by a multigene family.
Based on sequence and hybridization data, the zein multigene family is divided into several subfamilies. Each subfamily is defined by sequence homology to a cDNA clone: A20, A30, B49, B59, or B36. Hybrid-select translation studies which employ B49 and B36 select mRNAs that code for predominantly heavy class (23 kD) α-zein proteins, while A20, A30, and B59 select for predominantly the light class (19 kD) α-zein proteins (Heidecker and Messing, 1986). A comparison of zein sequences in each of the subfamilies A20, A30 and B49 have identified four distinct functional domains (Messing et al., 1983). Region I corresponds to the signal peptide present in most, if not all, zeins. Regions II and IV correspond to the amino and carboxyl termini, respectively, of the mature zein protein. Region III corresponds to the coding region between Regions II and IV, including a region which has tandem repeats of a 20 amino acid sequence.
There are several mutations known to cause reductions in zein synthesis that lead to alterations in the amino acid content of the seed. For example, in the seeds of plants homozygous for the recessive mutation opaque-2, the zein content is reduced by approximately 50% (Tsai et al., 1978). The opaque-2 mutation primarily affects synthesis of the 19 and 22 kD α-zein proteins, causing a significant decrease in the level of the 19 kD zein fraction and reducing the accumulation of the 22 kD zein fraction to barely detectable levels (Jones et al., 1977). In this mutant, there is a concomitant increase in the proportion of more nutritionally balanced proteins, e.g., albumins, globulins and glutelins, deposited in the seed. The net result of the altered storage protein patterns is an increase in the essential amino acids lysine and tryptophan in the mutant seed (Misra et al., 1972).
Two other recessive mutations,floury-2 and sugary-1, result in increased levels of methionine in the seed. The increased methionine content in the seeds of floury-2 mutants is the result of a decrease in the zein/glutelin ratio, due to reductions in the levels of both the 19 and 22 kD α-zein fractions, and an apparent increase in the methionine content of the glutelin fraction (Hansel et al., 1973; Jones, 1978). In sugary-1 mutants, there is a decrease in zein synthesis coupled with an increase in the methionine content of the zein and glutelin fractions (Paulis et al., 1978).
As demonstrated by the opaque-2,floury-2, and sugary-1 mutations, reductions in zein synthesis and/or changes in the relative proportions of the storage protein fractions can affect the overall amino acid composition of the seed. Unfortunately, poor agronomic characteristics (kernel softness, reduced yield, lowered resistance to disease) are associated with the opaque and floury mutations, preventing their ready application in commercial breeding.
Another way that genes can be down regulated in animals and plants involves the expression of antisense genes. A review of the use of antisense genes in manipulating gene expression in plants can be found in van der Krol et al. (1988a;1988b). The inhibition of expression of several endogenous plant genes has been reported. For example, U.S. Pat. No. 5,107,065 discloses down regulation of polygalacturonase activity by expression of an antisense gene. Other plant genes down regulated using antisense genes include the genes encoding chalcone synthase and the small subunit of ribulose-1,5-biphosphate carboxylase (van der Krol et al., 1988c; Rodermel et al., 1988). However, to date there has been no description of attempts to use antisense technology to alter the nutritional content of seeds.
Down regulation of gene expression in a plant may also occur through expression of a particular transgene. This type of down regulation is referred to as co-suppression and involves coordinate silencing of a transgene and a second transgene or a homologous endogenous gene (Matzke and Matzke, 1995). For example, cosuppression of a herbicide resistance gene in tobacco (Brandle et al., 1995), polygalacturonidase in tomato (Flavell, 1994) and chalcone synthase in petunia (U.S. Pat. No. 5,034,323) have been demonstrated. Flavell (1994) suggested that multicopy genes, or gene families, must have evolved to avoid cosuppression in order for multiple copies of related genes to be expressed in a plant.
Thus, there is a need for a method to alter the nutritional content of seeds and produce kernels with good agronomic characteristics, including maintaining kernel hardness, yield, and disease resistance of the parent genotype. Furthermore, there is a need for a method to decrease expression of seed storage proteins of poor nutritional quality while increasing proteins with higher contents of nutritionally advantageous amino acids, such as methionine and lysine, and/or while increasing the starch content of seeds.